Revision as of 17:40, 5 May 2007

This protocol is a variant of the Hanahan protocol [1] using CCMB80 buffer for DH10B, TOP10 and MachI strains. It builds on Example 2 of the Bloom05 patent as well. This protocol has been tested on TOP10, MachI and BL21(DE3) cells. See Bacterial Transformation for a more general discussion of other techniques. The Jesse '464 patent describes using this buffer for DH5α cells. The Bloom04 patent describes the use of essentially the same protocol for the Invitrogen Mach 1 cells.

Contents

Preparing glassware and media

Detergent is a major inhibitor of competent cell growth and transformation. Glass and plastic
must be detergent free for these protocols. The easiest way to do this is to avoid washing
glassware, and simply rinse it out. Autoclaving glassware filled 3/4 with DI water is an effective
way to remove most detergent residue. Media and buffers should be prepared in detergent free glassware
and cultures grown up in detergent free glassware.

Preparing seed stocks

streak TOP10 cells on an SOB plate and grow for single colonies at 23 C

room temperature works well

Pick single colonies into 2 ml of SOB medium and shake overnight at 23 C

room temperature works well

Add glycerol to 15%

Aliquot 1 ml samples to Nunc cryotubes

Place tubes into a zip lock bag, immerse bag into a dry ice/ethanol bath for 5 minutes

This step may not be necessary

Place in -80 freezer indefinitely.

Preparing competent cells

Inoculate 250 ml of SOB medium with 1 ml vial of seed stock and grow at 20 C to an OD of 0.3

This takes approximately 16 hours.

Controlling the temperature makes this a more reproducible process, but is not essential.

Room temperature will work. You can adjust this temperature somewhat to fit your schedule